Ras proteins are highly conserved guanine nucleotide binding enzymes that couple cell surface receptors to intracellular signaling pathways controlling cell proliferation and differentiation [Bourne et al., Nature, 349:117-127 (1991); Boguski and McCormick, Nature, 366:643-654 (1993)]. Ras proteins act as molecular switches by cycling between an active GTP-bound state and an inactive GDP-bound state. The nucleotide bound state of Ras is not determined by intrinsic equilibrium with cytoplasmic pools of guanine nucleotides but by the relative activities of two classes of regulatory proteins: GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). Exchange factors promote the activation of Ras by catalyzing exchange of GDP for GTP, whereas activating proteins control the conversion of Ras to the inactive state by stimulating the hydrolysis of GTP to GDP [Boguski and McCormick, Nature, 366:643-654 (1993)].
Cell surface receptors that signal via tyrosine kinases activate Ras by stimulating the guanine nucleotide exchange reaction [Medema et al., Molec. Cell. Biol., 13:155-162 (1993); Buday and Downward, Cell, 73:611-620 (1993); Gale et al., Nature, 363:88-92 (1993)]. Genetic and biochemical studies have indicated that this reaction is controlled by the Ras guanine nucleotide exchange factor Son of sevenless (Sos) [Bar-Sagi, Trends Endocrin. Metab., 5:165-169 (1994)]. Following ligand binding, Sos is recruited from the cytoplasm to the activated receptor in a phosphotyrosine-dependent manner through adapter proteins such as Grb2. Grb2 contains SH3 domains that are bound constitutively to a C-terminal proline-rich region of Sos, and the Grb2-Sos complex is recruited to activated receptors by interactions between the SH2 domain of Grb2 and phosphotyrosine residues on the receptor [Schlessinger, Trends Biochem. Sci., 18:273-275 (1994)]. Since Ras is localized to the membrane, receptor activation results in an increase in the effective concentration of Sos in the vicinity of Ras, thereby facilitating the exchange of bound guanine nucleotide for free cellular guanine nuzleotides. The cellular concentrations of GTP are .about.10 fold higher than that of GDP, and Sos-mediated guanine nucleotide exchange on Ras thus leads to transient accumulation of active GTP-bound Ras molecules.
Sos proteins are large (Mr.about.150 kD) and contain several functional domains [Chardin et al., Science, 260:1338-1343 (1993)]. They are expressed in a wide range of tissues, consistent with their role as activators of the ubiquitously expressed Ras genes. The region of Sos that is functional for nucleotide exchange on Ras spans about 500 residues, and contains blocks of sequence that are conserved in other Ras-specific nucleotide exchange factors such as Cdc25, Sdc25 and Ras guanine nucleotide release factor (GRF) [Boguski and McCormick, Nature, 366:643-654 (1993); Poullet et al., Eur. J. Biochem., 227:537-544 (1995)] (FIG. 1). Biochemical studies on these proteins have shown that the Ras-exchange factor complex is stable in the absence of nucleotides, and that the complex is dissociated by the re-binding of either GDP or GTP [Powers et al., Molec. and Cell. Biol., 9:390-395 (1989); Mistou et al., EMBO J., 11:2391-2397 (1992); Lai et al., Mol. Cell. Biol., 13:1345-1352 (1993); Haney and Broach, J. Biol. Chem., 269:16541-16548 (1994)]. The principal role for the exchange factor is to facilitate nucleotide release, and it does not appear to control the preferential rebinding of GTP over GDP to a significant extent [Haney and Broach, J. Biol. Chem., 269:16541-16548 (1994); Klebe et al., Biochemistry, 34:12543-12552 (1995)].
The utilization of nucleotide exchange to control the timing of critical molecular events is a mechanism that is common to many different cellular regulators. In addition to small guanine nucleotide binding proteins (G-proteins) homologous to Ras, such as the Arf, Rab, Rho, Rac and Ran, nucleotide exchange is also crucial to the timing cycles of the heterotrimeric G-proteins and ribosomal elongation factor Tu, which have catalytic cores that are structurally and functionally similar to Ras [Bourne et al., Nature, 349:117-127 (1991)]. Nucleotide exchange is also critical to the cycles of the protein chaperones of the DnaK/Hsp70 family, which utilize ATP to bind and release peptides and are unrelated in sequence or structure to the GTPases [Harrison et al., Science, 276:431435 (1997)].
In contrast to the high degree of structural conservation seen in the GTPases, there are distinct families of nucleotide exchange factors that are unrelated to each other. The structures of several small G-protein exchange factors have been determined in isolation, revealing a variety of protein architectures (Mss4 [Yu and Schreiber, Nature, 376:788-791 (1995)], ARNO/Sec7 [Mossessova et al., Cell, 92:415423 (1998); Cherfils et al., Nature, 392:101-105 (1998)] and RCC1 [Renault et al., Nature, 392:97-101 (1998)]). At the present time the structure of only one nucleotide exchange factor bound to its cognate guanine nucleotide binding proteins has been determined, that of EF-Tu bound to its exchange factor EF-Ts [Wang et al., Nat. Struct. Biol., 4:650-656 (1997); Kawashima et al., Nature, 379:511-518 (1996)]. In addition, the structure of the ATPase domain of DnaK bound to its exchange factor GrpE has also been determined [Harrison et al., Science, 276:431-435 (1997)]. No structural information is available on Ras-type small G-proteins complexed with their nucleotide exchange factors.
One means of modulating cellular proliferation and/or differentiation is to either inhibit or facilitate the Ras-Sos interaction. Therefore, there is a need to identify agonists or antagonists to the Ras-Sos complex. Unfortunately, such identification has heretofore relied on serendipity and/or systematic screening of large numbers of natural and synthetic compounds. A far superior method of drug-screening relies on structure based drug design. In this case, the three dimensional structure of Ras-Sos complex is determined and potential agonists and/or potential antagonists are designed with the aid of computer modeling [Bugg et al., Scientific American, Dec.:92-98 (1993); West et al., TIPS, 16:67-74 (1995); Dunbrack et al., Folding & Design, 2:27-42 (1997)]. However, heretofore the three-dimensional structure of the Ras-Sos complex has remained unknown. Therefore, there is a need for obtaining a crystal of a Ras-Sos complex with sufficient quality to allow high quality crystallographic data to be obtained. Furthermore there is a need for the determination of the three-dimensional structure of such crystals. Finally, there is a need for procedures for related structural based drug design predicated on such crystallographic data.
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